Cyclic AMP-dependent transcription factor-3 (ATF3), a stress sensor, plays an essential role in cells to maintain homeostasis and has diverse functions in cellular survival and death signal pathways. ATF3 is a novel regulator of p53 protein stability and function. The activities of ATF3 are modulated by post-translational modifications (PTMs), such as ubiquitination, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of this study was to investigate whether ATF3 is post-translationally modified by SUMO proteins and also to elucidate SUMOylation of ATF3 on TP53 gene activity. Here we report that ATF3 is clearly defined as a SUMO target protein both in vitro SUMOylation assay using recombinant proteins and at the cellular levels. Furthermore, ATF3 interacted with UBE2I, the only SUMO E2 enzyme found so far. In addition, PIAS3β (a SUMO E3 ligase) enhanced and SENP2 and SENP7 (two SUMOylation proteases) decreased SUMOylation of ATF3, respectively. Finally, we found that ATF3 is selectively SUMOylated at lysine residue 42 but the SUMOylation does not alter subcellular localization of ATF3. We then characterized the functional role of ATF3 SUMOylation on TP53 gene expression. We found that SUMOylation of ATF3 is required for full repression of TP53 gene. Overall, we provide the first evidence that ATF3 is post-translationally modified by SUMO and SUMOylation of ATF3 plays a functional role in regulation of TP53 gene activity. J. Cell. Biochem. 114: 589-598, 2013. © 2012 Wiley Periodicals, Inc.